On-line solid phase extraction/capillary reverse phase liquid chromatography has been regarded as a very important technological system in studying proteomes by virtue of its high efficiency in analysis. Particularly, this allows effective separation of trace amounts of biological substances and enables highly efficient identification of trace amounts of proteins due to its broad spectrum of analyte-solid phase reactions.
As a method for analyzing proteins, a mass spectrometry-based method has served as a standard analytic platform of proteome study. A typical example of the method, such as a shot-gun method or a bottom-up method, includes hydrolysis of proteins into peptides prior to the analysis using a mass spectrometer. Such hydrolysis increases the solubility of biological samples and produces peptide digests that may be ionized and detected easily in a mass spectrometer.
However, the above-mentioned method inevitably causes complexity of samples. For example, in the case of one of the simplest proteomes, yeast proteome, 300,000 or more peptide fragments are produced from about 6,000 diverse proteins. Therefore, in order to solve the complexity of samples, various methods including on-/off-line multidimensional protein identification technology have been developed (Link, A. J., Eng, J., Schieltz, D. M., Carmack, E., et al., Nat. Biotechnol. 1999, 17, 676-682; Chen, E. I., Newel, J., Felding-Habermann, B., Yates, J. R. III, Mol. Cell. Proteomics 2006, 5, 53-56). However, improvement in the efficiency and sensitivity of a liquid chromatography column is still required. In this context, it has been known that the sensitivity of analysis based on liquid chromatography/mass spectrometry may be increased rapidly when the inner diameter of a separation column is decreased while maintaining the length thereof (Kim, M.-S., Choie, W.-S., Shin, Y. S., Yu, M. H., Lee, S.-W., Bull. Korean Chem. Soc. 2004, 25, 1833-1839).
In addition, in the case of a biological sample containing a significant amount of detergents and salts, an on-line desalting operation is an essential process required prior to mass spectrometry. This is because such impurities interrupt ionization of the peptide sample to be analyzed, resulting in a drop in detection sensitivity for the peptide sample analysis. Thus, considering time saving and sample loss, on-line desalting is more efficient than off-line desalting. Further, when a capillary column having a large length and a small inner diameter is packed with hydrophobic material, a long period of time is required to accomplish equilibrium (or regeneration) of the column. For example, at least two hours is required for such equilibrium so that a column having a length of 1 m and an inner diameter of 75 μm may be reutilized.
Meanwhile, existing liquid chromatography devices have no solid phase extraction column, and thus require a long time for sample loading. Otherwise, even when such devices use a solid phase extraction column, their resolution is poor because the sample injecting direction is the same as the sample eluting direction.
In addition, existing liquid chromatography devices use an additional valve and/or pump in order to allow the sample injecting direction and the sample eluting direction to be opposite to each other (so-called back flushing). However, this approach makes the operation of a liquid chromatography device more complex and increases the possibility of malfunctioning of the device.